Method and system for detecting electrophysiological changes in pre-cancerous and cancerous tissue

ABSTRACT

A method and system are provided for determining a condition of a selected region of epithelial tissue. At least two current-passing electrodes are located in contact with a first surface of the selected region of the tissue. A plurality of measuring electrodes are located in contact with the first surface of the selected region of tissue as well. Electropotential and impedance are measured at one or more locations. An agent may be introduced into the region of tissue to enhance electrophysiological characteristics. The condition of the tissue is determined based on the electropotential and impedance profile at different depths of the epithelium, tissue, or organ, together with an estimate of the functional changes in the epithelium due to altered ion transport and electrophysiological properties of the tissue.

BACKGROUND OF INVENTION

[0001] The present invention relates generally to the detection ofabnormal or cancerous tissue and, more particularly, to the detection ofchanges in electrophysiological characteristics of abnormal or canceroustissue related to the functional, structural, and topographicrelationships of the tissue during the development of malignancy. Thesemeasurements may be made in the absence and/or presence ofpharmacological or hormonal agents to reveal and accentuateelectrophysiological characteristics indicative of abnormal or canceroustissue.

[0002] Cancer is a leading cause of death in both men and women in theUnited States. Difficulty in detecting abnormal pre-cancerous orcancerous tissue before treatment options become non-viable is onereason for the high mortality rate. Detecting the presence of abnormalor cancerous tissues is difficult, in part, because such tissues arelargely located deep within the body, thus requiring expensive, complex,invasive, and/or uncomfortable procedures. For this reason, the use ofdetection procedures is often restricted until a patient is experiencingsymptoms related to the abnormal tissue. Many forms of cancers ortumors, however, require extended periods of time to attain a detectablesize (and thus to produce significant symptoms or signs in the patient).It is often too late for effective treatment by the time the cancer ortumor is detected using currently available diagnostic modalities.

[0003] One proposed method for early detection of cancerous andpre-cancerous tissue includes measuring of the electrical impedance ofbiological tissue. For example, U.S. Pat. No. 3,949,736 discloses alow-level electric current passed through tissue, with a measurement ofthe voltage drop across the tissue providing an indirect indication ofthe overall tissue impedance. This method teaches that a change inimpedance of the tissue is associated with an abnormal condition of thecells composing the tissue, indicating a tumor, carcinoma, or otherabnormal biological condition. This disclosure, however, does notdiscuss either an increase or decrease in impedance associated withabnormal cells, nor does it specifically address tumor cells.

[0004] One disadvantage of this and similar systems is that the inherentDC electrical properties of the epithelium are not considered. Manycommon malignancies develop in an epithelium, often the cell layer thatlines a hollow organ, such as the bowel, or in ductal structures, suchas the breast or prostate. Epithelial tissue maintains a transepithelialelectropotential (TEP) that may be altered by the malignancy process.Early in the malignant process, the epithelium may lose itstransepithelial potential, particularly when compared to epithelium somedistance away from the developing malignancy. Thus, the combination oftransepithelial electropotential measurements with impedance may be moreaccurate in diagnosing pre-cancerous and cancerous conditions.

[0005] Another disadvantage of the above referenced system is that thefrequency range is not defined. Certain information may be obtainedabout cells according to the range of frequencies selected. Differentfrequency bands may be associated with different structural orfunctional aspects of the tissue. See, for example, F. A. Duck, PhysicalProperties of Tissues, London: Academic Press, 2001; K. R. Foster, H. P.Schwan, Dielectric properties of tissues and biological materials: acritical review, Crit. Rev. Biomed. Eng., 1989, 17(1): 25-104. Forexample at high frequencies, such as >1 GHz, molecular structure has adominating effect on the relaxation characteristics of the impedanceprofile. Relaxation characteristics include the delay in response of atissue to a change in the applied electric field. For example, anapplied AC current results in a voltage change across the tissue whichwill be delayed, or phase shifted, because of the impedancecharacteristics of the tissue. Relaxation and dispersion characteristicsof tissue vary according to the frequency of the applied signal.

[0006] At lower frequencies, such as <100 Hz, or the so calledα-dispersion range, alterations in ion transport and chargeaccumulations at large cell membrane interfaces dominate the relaxationcharacteristics of the impedance profile. In the frequency range betweena few kHz and 1 MHz, or the so-called β-dispersion range, cell structuredominates the relaxation characteristics of the epithelial impedanceprofile. Within this range at low kHz frequencies, most of the appliedcurrent passes between the cells through the paracellular pathway andtight junctions. At higher frequencies in the β-dispersion range thecurrent can penetrate the cell membrane and therefore passes bothbetween and through the cells, and the current density will depend onthe composition and volume of the cytoplasm and cell nucleus.

[0007] Characteristic alterations occur in the ion transport of anepithelium during the process of malignant transformation affecting theimpedance characteristics of the epithelium measured at frequencies inthe α-dispersion range. Later in the malignant process, structuralalterations with opening of the tight junctions and decreasingresistance of the paracellular pathways, together with changes in thecomposition and volume of the cell cytoplasm and nucleus, affect theimpedance measured in the β-dispersion range.

[0008] Another disadvantage of the above referenced system is that thetopography of altered impedance is not examined. By spacing themeasuring electrodes differently, the epithelium can be probed todifferent depths. The depth that is measured by two surface electrodesis approximately half the distance between the electrodes. Therefore,electrodes 1 mm apart will measure the impedance of the underlyingepithelium to a depth of approximately 500 microns. It is known, forexample, that the thickness of bowel epithelium increases at the edge ofa developing tumor to 1356±208 μ compared with 716±112μ in normal bowel.D. Kristt, et al. Patterns of proliferative changes in crypts borderingcolonic tumors: zonal histology and cell cycle marker expression.Pathol. Oncol. Res 1999; 5(4): 297-303. By comparing the measuredimpedance between electrodes spaced approximately 2.8 mm apart with theimpedance of electrodes spaced approximately 1.4 mm apart, informationabout the deeper and thickened epithelium may be obtained. See, forexample, L. Emtestam & S. Ollmar. Electrical impedance index in humanskin: measurements after occlusion, in 5 anatomical regions and in mildirritant contact dermatitis. Contact Dermatitis 1993; 28(2): 104-108.

[0009] Another disadvantage of the above referenced methods is that theydo not probe the specific conductive pathways that are altered duringthe malignant process. For example, potassium conductance is reduced inthe surface epithelium of the colon early in the malignant process.

[0010] Other patents, such as U.S. Pat. Nos. 4,955,383 and 5,099,844,disclose that surface electropotential measurements may be used todiagnose cancer. Empirical measurements, however, are difficult tointerpret and use in diagnosis. For example, the above referencedinventions diagnose cancer by measuring voltage differences(differentials) between one region of the breast and another and thencomparing them with measurements in the opposite breast. Changes in themeasured surface potential may be related to differences in theimpedance characteristics of the overlying skin. This fact is ignored bythe above referenced and similar inventions, resulting in a diagnosticaccuracy of 72% or less. J. Cuzick et al. Electropotential measurementsas a new diagnostic modality for breast cancer. Lancet 1998; 352(9125):359-363; M. Faupel et al. Electropotential evaluation as a new techniquefor diagnosing breast lesions. Eur. J. Radiol. 1997; 24 (1): 33-38.

[0011] Other inventions that use AC measurement, such as U.S. Pat. No.6,308,097, also have a lower accuracy than may be possible with acombination of DC potential measurements and AC impedance measurements.The above referenced system diagnoses cancer by only measuring decreasedimpedance (increased conductance) over a cancer.

[0012] Another potential source of information for the detection ofabnormal tissue is the measurement of transport alterations in themucosa. Epithelial cells line the surfaces of the body and act as abarrier to isolate the body from the outside world. Not only doepithelial cells serve to insulate the body, but they also modify thebody's environment by transporting salts, nutrients, and water acrossthe cell barrier while maintaining their own cytoplasmic environmentwithin fairly narrow limits. One mechanism by which the epithelial layerwithstands the constant battering is by continuous proliferation andreplacement of the barrier. This continued cell proliferation may partlyexplain why more than 80% of cancers are of epithelial cell origin.

[0013] It is known that the addition of serum to quiescent fibroblastsresults in rapid cell membrane depolarization. Cell membranedepolarization is an early event that may be associated with celldivision. Depolarization induced by growth factors appears biphasic insome instances, but cell division may be stimulated withoutdepolarization. Cell membrane depolarization is temporally associatedwith Na⁺ influx, and the influx persists after repolarization hasoccurred. Although the initial Na⁺ influx may result in depolarization,the increase in sodium transport may not cease once the cell membranehas been repolarized, possibly due to Na/K ATPase pump activation. Otherstudies also support that Na⁺ transport is altered during cellactivation. In addition to altered Na⁺ transport, transport of K⁺ and ofCl⁻ is altered during cell proliferation.

[0014] A number of studies have demonstrated that proliferating cellsare relatively depolarized when compared to those that are quiescent ornon-dividing. Differentiation is associated with the expression ofspecific ion channels. Additional studies indicate that cell membranedepolarization occurs because of alterations in ionic fluxes,intracellular ionic composition, and transport mechanisms that areassociated with cell proliferation.

[0015] Intracellular Ca²⁺ (Ca²⁺ _(i)) and intracellular pH (pH_(i)) areincreased by mitogen activation. Cell proliferation may be initiatedfollowing the activation of phosphatidylinositol which releases twosecond messengers, 1,2-diacylglycerol and inosotol-1,4,5-triphosphate,which trigger Ca²⁺ _(i) release from internal stores. Ca²⁺ _(i) andpH_(i) may then alter the gating of various ion channels in the cellmembrane, which are responsible for maintaining the voltage of the cellmembrane. Therefore, there is the potential for interaction betweenother intracellular messengers, ion transport mechanisms, and cellmembrane potential. Most studies have been performed in transformed andcultured cells and not in intact epithelia during the development ofcancer, so that it is largely unknown how up-regulated proliferationaffects cell membrane potential, transepithelial potential, epithelialimpedance, and ion transport during carcinogenesis.

[0016] It was known that cancer cells are relatively depolarizedcompared to non-transformed cells. It has been suggested that sustainedcell membrane depolarization results in continuous cellularproliferation and that malignant transformation results as a consequenceof sustained depolarization and a failure of the cell to repolarizeafter cell division. C. D. Cone Jr., Unified theory on the basicmechanism of normal mitotic control and oncogenesis. J. Theor. Biol.1971; 30(1): 151-181; C. D. Cone Jr., C. M. Cone. Induction of mitosisin mature neurons in central nervous system by sustained depolarization.Science 1976; 192 (4235): 155-158; C. D. Cone, Jr. The role of thesurface electrical transmembrane potential in normal and malignantmitogenesis. Ann. N.Y. Acad. Sci. 1974; 238: 420-435. A number ofstudies have demonstrated that cell membrane depolarization occursduring transformation and carcinogenesis. Other studies havedemonstrated that a single ras-mutation may result in altered iontransport and cell membrane depolarization. Y. Huang, S. G. Rane, Singlechannel study of a Ca(2+)-activated K+ current associated with rasinduced cell transformation. J. Physiol. 1993; 461: 601-618. Forexample, there is a progressive depolarization of the colonocyte cellmembrane during 1,2 dimethylhydrazine (DMH)-induced colon cancer in CF₁mice. The V_(A) (apical membrane voltage) measured with intracellularmicroelectrodes in histologically “normal” colonic epitheliumdepolarized from −74.9 mV to −61.4 mV after 6 weeks of DMH treatment andto −34 mV by 20 weeks of treatment.

[0017] While epithelial cells normally maintain their intracellularsodium concentration within a narrow range, electronmicroprobe analysisshows that cancer cells exhibit cytoplasmic sodium/potassium ratios thatare three to five times greater than those found in theirnon-transformed ones. These observations partly explain the electricaldepolarization observed in malignant or pre-malignant tissues, becauseof the loss of K⁺ or Na⁺ gradients across the cell membrane.

[0018] In addition to cell membrane depolarization, and alteredintracellular ionic activity, other studies have shown that there may bea decrease in electrogenic sodium transport and activation ofnon-electrogenic transporters during the development of epithelialmalignancies. These changes may occur as a consequence of alteredintracellular ionic composition. Other specific ion transportalterations have been described in colon, prostate, breast, uterinecervix, melanoma, urothelium, and pancreas during proliferation,differentiation, apoptosis, and carcinogenesis.

[0019] Apoptosis or physiological cell death is down-regulated duringthe development of malignancy. Ion transport mechanisms affected byapoptosis include the influx of Ca²⁺, non-selective Ca²⁺-permeablecation channels, calcium-activated chloride channels, andK⁺-Cl⁻-cotransport. J. A. Kim et al. Involvement of Ca2+ influx in themechanism of tamoxifen-induced apoptosis in Hep2G human hepatoblastomacells. Cancer Lett. 1999; 147(1-2): 115-123; A. A. Gutierrez et al.Activation of a Ca2+-permeable cation channel by two different inducersof apoptosis in a human prostatic cancer cell line. J. Physiol. 1999;517 (Pt. 1): 95-107; J. V. Tapia-Vieyra, J. Mas-Oliva. Apoptosis andcell death channels in prostate cancer. Arch. Med. Res. 2001; 32(3):175-185; R. C. Elble, B. U. Pauli. Tumor Suprression by a ProapoptoticCalcium-Activated Chloride Channel in Mammary Epithelium. J. Biol. Chem.2001; 276(44): 40510-40517.

[0020] Loss of cell-to-cell communication occurs during carcinogenesis.This results in defective electrical coupling between cells, which ismediated via ions and small molecules through gap junctions, which inturn influences the electrical properties of epithelia.

[0021] Polyps or overtly malignant lesions may develop in a backgroundof disordered proliferation and altered transepithelial ion transport.Experimental animal studies of large bowel cancer have demonstrated thattransepithelial depolarization is an early feature of the pre-malignantstate. In nasal polyp studies, the lesions had a higher transepithelialpotential, but these lesions were not pre-malignant in the same sense asan adenomatous or pre-malignant colonic polyp, that are usuallydepolarized. Electrical depolarization has been found in biopsies ofmalignant breast tissue. Recently alterations in impedance have beenfound to be associated with the pre-malignant or cancerous state inbreast and bowel.

[0022] DC electrical potential alterations have been reported to beuseful to diagnose non-malignant conditions such as cystic fibrosis,cancer in animal models, human cells or isolated tissue, and in man.Differences in impedance between normal tissue and cancer have beendescribed in animal models in vitro and have been applied to in vivocancer diagnosis. DC potential measurements have not been combined withimpedance measurements to diagnose cancer, however, becauseelectrophysiological alterations that accompany the development ofcancer are generally not fully characterized. Transepithelialdepolarization is an early event during carcinogenesis, which may affecta significant region of the epithelium (a “field defect”). Thisdepolarization is accompanied by functional changes in the epitheliumincluding ion transport and impedance alterations. Early on in theprocess these take the form of increased impedance because of decreasedspecific electrogenic ion transport processes. As the tumor begins todevelop in the pre-malignant epithelium, structural changes occur in thetransformed cells such as a breakdown in tight junctions and nuclearatypia. The structural changes result in a marked reduction in theimpedance of the tumor. The pattern and gradient of electrical changesin the epithelium permit the diagnosis of cancer from a combination ofDC electrical and impedance measurements. Another reason that DCelectropotential and impedance measurements have not been successfullyapplied to cancer diagnosis is that transepithelial potential andimpedance may be quite variable and are affected by the hydration state,dietary salt intake, diurnal or cyclical variation in hormonal level, ornon-specific inflammatory changes and other factors. In the absence ofknowledge about the physiological variables which influencetransepithelial potential and impedance these kinds of measurements maynot be reliable to diagnose pre-malignancy or cancer. Furthermore adetailed understanding of the functional and morphological alterationsthat occur during carcinogenesis permits appropriate electrical probingfor a specifically identified ion transport change that is alteredduring cancer development. For example knowledge that electrogenicsodium absorption is reduced during cancer development in the colonpermits the use of sodium channel blockers (e.g., amiloride) or varyingsodium concentration in the ECM to examine whether there is aninhibitable component of sodium conductance. By varying the depth of themeasurement (by measuring the voltage drop across differently spaceelectrodes), it is possible to obtain topographic and depth informationabout the cancerous changes in the epithelium.

[0023] The diagnostic accuracy of current technology using DCelectropotentials or impedance alone has significant limitations.Sensitivity and specificity for DC electrical measurements in the breasthave been reported as 90% and 55% respectively and 93% and 65% forimpedance measurements. This would result in an overall diagnosticaccuracy of between 72-79%, which is probably too low to result inwidespread adoption. J. Cuzick et al. Electropotential measurements as anew diagnostic modality for breast cancer. Lancet 1998; 352 (9125):359-363; A. Malich et al. Electrical impedance scanning for classifyingsuspicious breast lesions: first results. Eur. Radiol. 2000; 10(10):1555-1561. The combination of DC electrical potentials and impedancespectroscopy may result in a diagnostic accuracy of greater than 90%which will lead to improved clinical utility.

[0024] Thus, there remains a need for effective, practical methods ofdetecting abnormal tissue.

SUMMARY OF THE INVENTION

[0025] To overcome problems and inadequacies associated with priormethods, abnormal or cancerous tissue is characterized using DCmeasurements and impedance measurements in combination. DC measurementsprovide information about the functional state of the epithelium and candetect early pre-malignant changes and an adjacent malignancy. Impedancemeasurements at different frequencies using differently spacedelectrodes provide depth and topographic information to give bothstructural (high frequency range) and functional (low frequency range)information about the tissue being probed. Abnormal or cancerous tissuecan be detected and characterized by detecting and measuring transportalterations in mucosal tissues, using ionic substitutions and/orpharmacological and hormonal manipulations to determine the presence ofabnormal pre-cancerous or cancerous cells. A baseline level oftransepithelial DC potential, impedance, or other electrophysiologicalproperty that is sensitive to alterations in transport in epithelia ismeasured in the tissue to be evaluated. An agent may be introduced toenhance the transport or make it possible to detect the transportalteration. The transepithelial DC potential and/or impedance of thetissue (or other electrophysiological property that may reflect or makeit possible to detect alterations in the transport) are then measured.Based on the agent introduced and the measured electrophysiologicalparameter, the condition of the tissue is determined.

[0026] A method and system are provided for determining a condition of aselected region of epithelial tissue. At least two current-passingelectrodes are located in proximity to or in contact with a firstsurface of the selected region of the tissue. Alternatively, the currentpassing electrodes may pass current across the tissue or epithelium. Forexample, current may be passed between the urethra and surface of theprostate, accessed per rectum; between the abdominal wall and the bowelmucosal surface; between the skin surface of the breast and the centralbreast ducts accessed by central duct catheter or ductoscope. Aplurality of measuring electrodes is located in contact with or inproximity with the first surface of the selected region of tissue aswell. A signal is established between the current-passing electrodes.One or more of the measuring electrodes measures impedance associatedwith the established signal. Alternatively a three electrode system maybe used for measurements whereby one electrode is used for both currentinjection and voltage recording. An agent is introduced into the regionof tissue. The condition of the tissue is determined based on the effectof the agent on measured DC transepithelial potential impedance or otherelectrophysiological characteristics. The electrodes in the describedmethods and apparatus can be used in contact with, in proximity to,over, or inserted into the tissue being examined. It should beunderstood that where the method is described in an embodiment asencompassing one of these arrangements, it is contemplated that it canalso be used interchangeably with the other. For example, where themethod is described as having an electrode in contact with the tissue,the method can also be used with the electrode inserted into or inproximity to the tissue. Similarly, where the method is described ashaving an electrode in proximity to the tissue, it is contemplated thatthe electrode can also be in contact with or inserted into the tissue.

[0027] In order to more accurately detect transport alterations inabnormal pre-cancerous or cancerous epithelial tissue, a pharmacologicalagent may be introduced to manipulate the tissue. Pharmacological agentsmay include agonists of specific ion transport and electrical activity,antagonists of specific ion transport and electrical activity, ionicsubstitutions, and/or hormonal or growth factor stimulation orinhibition of electrical activity.

[0028] Depending on the location of the tissue to be investigated, anumber of methods may be used to administer the pharmacological orhormonal agents. One exemplary method includes introducing the agentdirectly to the tissue being investigated, via either direct contact orinjection. Another exemplary method includes applying the agent to theskin surface, wherein the agent acts transcutaneously, or through theskin. Yet another exemplary method includes electroporation, wherein theepithelium or surface is made permeable by the passage of alternatingcurrent via electrodes in contact or penetrating the organ or epitheliumof interest. The agent then passive diffuses into the organ and itsconstituent cells. Additional exemplary methods include via inhalation,oral administration, lavage, gavage, enema, parenteral injection into avein or artery, sublingually or via the buccal mucosa, or viaintraperitoneal administration. One skilled in the art will appreciatethat other methods are possible and that the method chosen is determinedby the tissue to be investigated.

[0029] Thus, systems and methods consistent with the present inventionuse a combination of transepithelial electropotential and impedancemeasurements to diagnose pre-malignancy or cancer. Further, systems andmethods consistent with the present invention use a defined set offrequencies in combination to characterize functional and structuralalterations in pre-malignancy and cancer. By using spaced electrodes thepresent invention may provide topographic and geometrical (depth)information about the epithelium under examination to diagnosepre-malignancy and cancer. In one embodiment, systems and methods of thepresent invention use electrodes with specially formulated ECMs toprovide functional information about the epithelium to diagnosepre-malignacy and cancer.

[0030] Additional objects and advantages of the invention will be setforth in part in the description which follows, and in part will beobvious from the description, or may be learned by practice of theinvention. The objects and advantages of the invention will be realizedand attained by means of the elements and combinations particularlypointed out in the appended claims.

[0031] It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings, which are incorporated in andconstitute a part of this specification, illustrate one embodiment ofthe invention and together with the description, serve to explain theprinciples of the invention. In the Figures:

[0033]FIG. 1 is a schematic diagram of a DC and AC impedance measuringdevice, consistent with an embodiment of the present invention;

[0034]FIG. 2 illustrates an exemplary embodiment of a device suitablefor use with systems and methods consistent with the present invention;

[0035]FIG. 3 illustrates another exemplary embodiment of a devicesuitable for use with systems and methods consistent with the presentinvention;

[0036]FIGS. 4A and 4B illustrates other exemplary embodiments of adevice suitable for use with systems and methods consistent with thepresent invention;

[0037]FIGS. 5A and 5B illustrate the short circuit current associatedwith human colonic epithelium ex-vivo;

[0038]FIG. 6 is a photomicrograph illustrating electrophysiologic andhistologic alterations that may be present in colonic cancer;

[0039]FIG. 7 illustrates measurements of epithelial electropotential ina patient with rectal cancer;

[0040]FIG. 8 illustrates varying ionic content and the effect ontransepithelial conductance in human breast epithelium;

[0041]FIG. 9 illustrates measurements of cell membrane potential inhuman breast epithelial cells;

[0042]FIG. 10 illustrates the effect of increasing estradiol on thetransepithelial potential in benign and malignant breast epithelia;

[0043]FIG. 11 illustrates conductance and electropotential measurementsmade over the surface of the breast in women with and without breastcancer;

[0044]FIG. 12 illustrates the measurement of electropotential at thesurface of the breast, and variation of the measurement during menstrualcycle ;

[0045]FIG. 13 illustrates measurements of cell membrane potential inhuman prostatic epithelial cell under different growth conditions;

[0046]FIG. 14 illustrates measurements of electropotential in a patientwith normal prostate; and

[0047]FIG. 15 illustrates measurements of electropotential in a patientwith prostate cancer.

DETAILED DESCRIPTION

[0048] Reference will now be made in detail to an embodiment of theinvention, an example of which is illustrated in the accompanyingdrawings. Wherever possible, the same reference numbers will be usedthroughout the drawings to refer to the same or like parts.

[0049] In order to combine DC transepithelial measurement with impedancemeasurements, it may be necessary to obtain baseline measurement of theDC potential using the voltage sensing electrodes, referenced to a lowimpedance surface electrode, or the blood stream via an IV, or theinterstitial body fluid via a needle electrode or electrode thatpermeabilizes the overlying epidermis or other epithelium, or other bodyreference point. The electrodes may contain different ionicconcentrations, pharmacological agents, or hormones in their ECMs. Asused in this description, an ECM is a medium that permits transmissionof electrical signals between the surface being measured and theelectrode. An agent includes any ionic concentration, pharmacologicalagent, hormone, or other compound added to the ECM or otherwiseintroduced to the tissue under investigation, selected to providefurther information about the condition of the tissue. In anotherembodiment the concentrations of agents may be changed using a flowthrough system.

[0050] In order to measure the depth of the impedance alteration, avoltage drop is made between electrodes with different spacing. Spacingis determined by knowledge of the depth to be probed. Similarly, twodifferent frequency ranges will be used to measure functional andstructural changes at different depths.

[0051] In order to more accurately detect the functional transportalterations at different depths in abnormal pre-cancerous or cancerousepithelial tissue, an agent, such as a pharmacological agent, mayintroduced to manipulate the tissue, while electrically probing thetissue at different frequencies and monitoring the voltage drop betweendifferently spaced electrodes. Pharmacological agents include agonistsof specific ion transport and electrical activity, antagonists ofspecific ion transport and electrical activity, ionic substitutions,and/or hormonal or growth factor stimulation, which modulates, inhibitsor stimulates electrical activity.

[0052] Depending on the location of the tissue to be investigated, anumber of methods may be used to administer the pharmacological orhormonal agents. One exemplary method includes introducing the agentdirectly to the tissue being investigated, via either direct contact orinjection. Another exemplary method includes applying the agent to theskin surface, wherein the agent acts transcutaneously, or through theskin. Another exemplary method includes electroporation, wherein theepithelium or surface is made permeable by the passage of alternatingcurrent via electrodes in contact with or penetrating the organ orepithelium of interest. The agent then passively diffuses into the organand its constituent cells. Additional exemplary methods include viainhalation, oral administration, lavage, gavage, enema, parenteralinjection into a vein or artery, sublingually or via the buccal mucosa,or via intraperitoneal administration. One skilled in the art willappreciate that other methods are possible and that the method chosen isdetermined by the tissue to be investigated.

[0053] Based on the agent introduced and the tissue being investigated,measurements of electrophysiological properties, such as impedance, areperformed. Other properties that can be measured includes,transepithelial potential, changes in spontaneous oscillations intransepithelial potential or impedance associated with the malignantstate, and time delay in a propagation signal between electrodes, whichindicates a change or loss of gap-junction function. The results ofthese measurements are then used to determine the condition of theinvestigated tissue. For example, research has indicated that specificion transport processes are altered during the development of cancer.For example, a loss of electrogenic Na⁺ transport, an up-regulation inNa/H exchange, a down-regulation in K⁺ conductance, a decrease in basalCl⁻ absorption, and a down-regulation in c-AMP (cyclicadenosine-3′,5′-cyclic monophosphate) stimulated Cl⁻ secretion have beenobserved.

[0054] Thus, by administering agents appropriate to the particularepithelial tissue and measuring the associated electrophysiologicalcharacteristics, it is possible to detect abnormal pre-cancerous orcancerous tissue while the development of such tissue is at an earlystage. The method and system of the present invention is applicable toany epithelial derived cancer, such as, but not limited to, prostate,colon, breast, esophageal, and nasopharyngeal cancers, as well as otherepithelial malignancies, such as lung, gastric, uterine cervix,endometrial, skin, and bladder.

[0055] Specifically, in cancers affecting mucosal or epithelial tissues,transport alterations may be sufficiently large to suggest that they area consequence of an early mutation, affecting a large number of cells(i.e., a field defect). In this case, they may be exploited as potentialbiomarkers for determining which patients should be either morefrequently monitored, or conversely, may be used to identify particularregions of mucosa that require biopsy. The latter is especially helpfulin the case of flat adenomas or dysplasia, which are more difficult todetect physically than, for example, polyps.

[0056] A number of variations are possible for devices to be used withthe present invention. Further, within a device design, there are anumber of aspects that may be varied. These variations, and others, aredescribed below.

[0057] One probe or other device includes a plurality of miniaturizedelectrodes in recessed wells. Disposable commercially available siliconchips processing, such as filtering, may perform surface recording andinitial electronic processing. Each ECM solution or agent may bespecific to the individual electrode and reservoir on the chip. Thus,for one measurement, a particular set of electrodes is used. For anothermeasurement, for example, at a different ionic concentration, adifferent set of electrodes is used. While this produces somevariations, as the electrodes for one measurement are not located at thesame points as for another, this system provides generally reliableresults.

[0058] An alternative approach is to use fewer electrodes and use aflow-through or microfluidic system to change solutions and agents.Specifically, solutions or agents are changed by passing small amountsof electrical current to move solution or agent through channels and outthrough pores in the surface of the probe. In this embodiment, theelectrode remains in contact with the same region of the epithelium,thus eliminating region-to-region variation in measurement. Thisapproach requires time for equilibration between different solutions.

[0059] In detecting the presence of abnormal pre-cancerous or cancerousbreast tissue, a hand-held probe is provided for obtaining surfacemeasurements at the skin. The probe may include electrodes for passingcurrent as well as for measuring. An impedance measurement may be takenbetween the nipple cup electrode and the hand-held probe, or may betaken between electrodes on the hand-held probe. After taking initial DCmeasurements, a wetting/permeabilizing agent may be introduced to reduceskin impedance. The agent may be introduced using a microfluidicapproach, as described above, to move fluid to the surface of theelectrodes. Alternatively, surface electrodes that just penetrate thestratum corneum may be used to decrease impedance.

[0060] Regardless of the configuration of the device, FIG. 1 is aschematic of a DC and AC impedance measurement system 100 used in cancerdiagnosis, consistent with the present invention. The system 100interfaces with a probe device 105 including multiple electrodes,wherein the actual implementation of the probe device 105 depends on theorgan and condition under test. The probe device 105 may incorporate theelectrodes attached to a glove, needle, body cavity, endoscopic, orsurface probe. A reference probe 110 may take the form of an intravenousprobe, skin surface probe, or epithelial surface reference probedepending on the test situation and organ under investigation.

[0061] To avoid stray capacitances, the electrodes may be connected viashielded wires to a selection switch 120 which may select a specificprobe 105 following a command from the Digital Signal Processor (DSP)130. The selection switch 120 also selects the appropriate filterinterfaced to the probe 105, such that a low pass filter is used duringDC measurements and/or an intermediate or high pass filter is usedduring the AC impedance measurements. The selection switch 120 passesthe current to an amplifier array 140 which may be comprised of multipleamplifiers or switch the signals from different electrodes through thesame amplifiers when multiple electrodes are employed. In a preferredembodiment digital or analogue lock-in amplifiers are used to detectminute signals buried in noise. This enables the measurement of thesignal of interest as an amplitude modulation on a reference frequency.The switching element may average, sample, or select the signal ofinterest depending on the context of the measurement. This processing ofthe signal will be controlled by the DSP following commands from theCPU. The signals then pass to a multiplexer 150, and are serializedbefore conversion from an analogue to a digital signal by the ADC. Aprogrammable gain amplifier 160 matches the input signal to the range ofthe ADC 170. The output of the ADC 170 passes to the DSP 130. The DSP130 processes the information to calculate the DC potential and itspattern on the epithelial or skin surface as well as over the region ofsuspicion. In addition the impedance at varying depth and response ofthe DC potential and impedance to different ECM concentrations of ions,drug, hormones, or other agent are used to estimate the probability ofcancer. The results are then sent to the CPU 180 to give a test result185.

[0062] Alternatively the signal interpretation may partly or completelytake place in the CPU 180. An arbitrary waveform generator 190 or sinewave frequency generator will be used to send a composite waveformsignal to the probe electrodes and tissue under test. The measuredsignal response (in the case of the composite wave form stimulus) may bedeconvolved using FFT (Fast Fourier Transforms) in the DSP 130 or CPU180 from which the impedance profile is measured under the differenttest conditions. An internal calibration reference 195 is used forinternal calibration of the system for impedance measurements. DCcalibration may be performed externally, calibrating the probe beingutilized against an external reference electrolyte solution.

[0063]FIG. 2 illustrates a glove that may be used, for example, indiagnosis of prostate cancer or as a screening test for colorectalneoplasia. Multiple sensor electrode arrays may be attached to anexamining glove together with current passing electrodes. The individualelectrodes may be recessed and ECMs with different composition may beused to pharmacologically, electrophysiologically, or hormonally probethe epithelium under test. Spacing of the electrodes may be greater forthe prostate configuration than for other organ systems so that deepertissue may be electrically probed and the impedance of the deeper tissueevaluated. The electrodes will be interfaced via electrical wire, orwireless technology, with the device described in FIG. 1 above.

[0064]FIG. 3 is a schematic of an endoscopic probe, consistent with thepresent invention, which may be placed in contact with the epitheliumendoscopically. This probe may either be placed passively in contactwith the epithelium or held in place by pneumatic suction over theregion of interest. Ports are in place for the exchange of solutions orfor fluid exchange and suction. Guard rings may be incorporated toprevent cross-talk between electrodes and to force current from thecontact surface into the epithelium. In this configuration there arefour current passing electrodes each positioned radially 90° apart. Thispermits current to be passed and the voltage response to be measured inperpendicular fields. This enables the effects of surface asymmetry onimpedance (such as occurs with aberrant crypt foci) to be measured.Electrodes may be slightly recessed so as not to influence currentdensity measured at the surface.

[0065]FIG. 4A includes a handheld probe 400, consistent with the presentinvention, which may be applied to the surface of the breast. The probemay include a handle 410. The probe 400 may be attached, either directlyor indirectly using, for example, wireless technology, to a measurementdevice 420. The probe 400 may be referenced to an intravenous electrode,a skin surface electrode, or other ground. In one embodiment,illustrated in FIG. 4A, the reference is a nipple electrode or ductalprobe 430, illustrated in greater detail at close-up 440. One advantageof this configuration is that DC electropotential and impedance can bemeasured between the nipple electrode 430 and the probe 400. Themeasurement is thus a combination of the DC potentials and impedance ofthe breast ductal epithelium, non-ductal breast parenchyma, and theskin.

[0066] Referring to close-up 440, the ductal probe is inserted into oneof several ductal orifices that open onto the surface of the nipple.Ductal probe 443 is shown within a ductal sinus 444, which drains alarger collecting duct 445.

[0067] Another advantage of using a nipple electrode is that a solutionfor irrigating the ductal system may be exchanged through the probe,permitting introduction of pharmacological and/or hormonal agents. Asshown in magnified nipple probe 443, 443′ fluid can be exchanged througha side port. Fluid may be infused into the duct and aspirated at theproximal end (away from the nipple) of the nipple probe. Differentelectrolyte solutions may be infused into the duct to measure alteredpermeability of the ductal epithelium to specific ions or the epitheliummay be probed with different drugs to identify regions of abnormality.Estradiol, or other hormonal agents, may be infused into a breast ductto measure the abnormal electrical response associated withpre-malignant or malignant changes in the epithelium.

[0068] It should be understood that different configurations may also beused, such as a modified Sartorius cup that applies suction to thenipple. With this configuration, gentle suction is applied to a cupplaced over the nipple. Small amounts of fluid within the large ductsand duct sinues make contact with the electrolyte solution within theSartorius cup, establishing electrical contact with the fluid fillingthe breast ducts. DC or AC measurements may then be made between the cupand a surface breast probe.

[0069]FIG. 4B illustrates the probe 400 of FIG. 4A in greater detail.The skin contact of the surface 450 is placed in contact with thebreast. The surface electrodes 451 measure DC or AC voltages. Thecurrent passing electrodes 452 are used for impedance measurements.Probe 400 may also include one or more recessed wells containing one ormore ECMs.

[0070] Further embodiments of this technique may involve the use ofspaced electrodes to probe different depths of the breast, and the useof hormones, drugs, and other agents to differentially alter theimpedance and transepithelial potential from benign and malignant breasttissue, measured at the skin surface. This enables further improvementsin diagnostic accuracy.

EXAMPLE 1

[0071] Colon Cancer

[0072] In colon cancer, the following electrophysiological changes havebeen observed during the development of the abnormal tissue: loss ofelectrogenic Na+ transport, up-regulation in Na/H exchange,down-regulation in K⁺ conductance, decrease in basal Cl⁻ absorption, anddown-regulation in c-AMP (cyclic adenosine-3′,5′-cyclic monophosphate)stimulated Cl⁻ secretion. A number of pharmacological and hormonalmanipulations can be performed to detect these ion transportalterations.

[0073] By using electrolyte conductive medium (ECM) of differentconcentrations, the conductance of specific ions can be estimated andthe response to different pharmacological probes can be determined.Different pharmacological agents are administered that influenceelectrophysiological properties of normal bowel, but have minimal ordifferent effects on pre-cancerous or cancerous tissue. For example,glucocorticoids or mineralocorticoids, administered by injection ororally, increase the transepithelial electropotential (TEP) of normalcolon, but have a lesser effect on pre-cancerous or cancerous tissue.These steroids up-regulate electrogenic sodium absorption, therebydecreasing sodium specific impedance in normal colon.

[0074] The measured TEP decreases in response to a topically appliedamiloride (a sodium channel blocker) in normal colonic mucosa. Thisresponse is reduced by approximately 50% in pre-cancerous mucosa or bygreater than 75% in cancerous mucosa. In addition, the loss in sodiumconductance results in an increase of impedance of the surfaceepithelium. This ion transport alteration may be measured by determiningthe change in TEP as well as the basal impedance. In abnormalpre-cancerous or cancerous tissue, the TEP is lower, the response of theTEP to amiloride is less, and the increase in impedance (observed innormal colon in response to amiloride) is less in abnormal pre-cancerousor cancerous tissue. Similar pharmacological agents may be introducedthat alter the effect of chloride or potassium ion transport, whichaffect abnormal pre-cancerous or cancerous tissue in a different mannerthan in normal colon tissue.

[0075] It is important to note that the impedance is higher, orconductance is generally lower, around the edge of the tumor or in theimmediately adjacent pre-malignant epithelium. At more than 5-10 cm fromthe tumor the TEP is lower and ion specific impedances may be higher. Inthe tumor itself the impedance is lower (conductance higher).Measurement may be made over a suspected tumor, but also adjacent andsome distance away from the suspected tumor to more accurately identifythe cancerous or pre-cancerous tissue. There are also pharmacologicaldifferences between normal pre-cancerous and cancer tissue. Directcomparison between these different regions can used to make a moreaccurate diagnosis of cancer or premalignancy.

[0076] In one embodiment, electrophysiological measurements areperformed using a series of two or more electrodes attached to anexamining glove or probe. Some factors influencing the spacing of theelectrode and the signal used include the depth of penetration desiredand permeabilization of the surface epithelium using penetrating agents.A probe that permits variable frequency signals and varying electrodeplacement provides the most versatile arrangement, but a probe or gloveproviding a single frequency signal and/or static electrode placementmay also be used.

[0077] Sodium: Sodium conductance and absorptive properties in thesurface cells of the colonic epithelium are markedly attenuated in somepre-cancerous and cancerous cells. By measuring the impedance of thecolonic epithelium using low frequency sine waves and closely placedelectrodes, it is possible to determine the electrophysiologicalactivity of the surface cells. Passive electrodes, placed betweencurrent-passing electrodes, measure the impedance, while ECMs ofdifferent sodium concentration may be used to reveal alterations of thespecific ionic permeabilities of the epithelium. By using higherfrequency sine waves and widely spaced electrodes and ECMs of varyingsodium concentration, it is possible to estimate overall andion-specific conductances of the deeper epithelium. A ratio may bedetermined, expressed as the change in surface to deep sodiumconductance. The surface/deep sodium conductance ratio progressivelydecreases as tissue develops from at-risk, to pre-cancerous to canceroustissue. The surface cells that are conductive to sodium are replaced bycells from the deeper epithelium that do not have as high a conductance.Therefore, the ratio of surface Na⁺ conductance/deep Na⁺ conductancegoes from >2.0 to <1.0. Both the ratio and absolute number change.Measuring the ratio effectively normalizes the measurement for theparticular individual and epithelial region under test.

[0078] A number of ECMs and pharmacological agents may be employed tocharacterize the sodium transport characteristics of colonic tissue. Inone exemplary method, initial measurements are made using an electrolytesolution containing 10 mM KCl in the ECM, either in gel or solution,which interfaces between the electrode and the bowel wall. Measurementsare taken relative to an intravenous reference electrode or a lowimpedance skin electrode, having a minimal offset voltage relative tothe underlying extracellular fluid and bloodstream. The TEP is thenmeasured at increasing levels of sodium, both in the absence andpresence of amiloride or similar agent, such as benzamil, (10 μM-1 mM)to block electrogenic sodium transport. The difference between the twomeasurements will be the TEP attributable to the electrogenic sodiumtransport across the bowel epithelium. The electrogenic component ofsodium transport is diminished by 40-50% in colonic epithelium that isat-risk or pre-cancerous.

[0079] One method for varying the sodium and/or pharmacological contentduring measurement include using one or more wells or reservoirsassociated with each electrode, containing different concentrations ofelectrolyte and/or agent, so that the solution is not actually changedduring measurement but the measurement occurs under different conditionswith different electrodes and ECMs. Another method involves aflow-through solution change system, whereby solution changes may beautomated while using fewer electrodes.

[0080] Potassium: Measurements similar to that described above, withreference to sodium, are performed with reference to potassium.Specifically, an early decrease in potassium conductance is associatedwith at-risk or pre-cancerous colonic epithelium. As cancer develops,potassium conductance becomes up regulated and potassium secretion maybe enhanced. The decrease, and then increase, in potassium conductanceenables not only identification of abnormal tissue, but also thedetermination of the condition of the tissue, as either normal, at-risk,pre-cancerous, or cancerous.

[0081] Impedance measurements may be performed at varying concentrationsof potassium, using signals of varying frequency, and using variablyspaced electrodes, thus providing an impedance profile including thesuperficial and deep epithelium. For example, one method of determiningan impedance profile, with reference to potassium, is as follows: A TEPmeasurement is made using increasing concentrations of K⁺ and allmeasurements are performed using ECM containing amiloride or anotherblocker of the electrogenic Na⁺ pump to remove the contribution ofelectrogenic Na⁺ transport to TEP. Using the well method describedabove, the ECM in each well contains a combination of amiloride,bethanacol, forskolin, and 3-isobutyl-1-methylxanthine (IBMX). Each ofthe four wells contains varying K⁺ concentrations (between 10 and 80mM), while maintaining the concentration of Na and Cl ions. These agents(bethanacol, forkskolin, and IBMX) depolarize the cell membrane bymaximally opening Cl⁻ conduction channels in the surface cells of thecolon. This cell membrane depolarization results in the opening ofvoltage-sensitive K+ channels in the cell membrane. Specifically,bethanacol (or carbacol) raises intracellular Ca²⁺ which opens Ca²⁺sensitive K⁺ channels, as well as increasing chloride secretion openingup Cl⁻ channels. Other muscarinic agonists may produce similar results.Forkskolin increases adenyl cyclase, thereby raising intracellular c-AMPopening up K⁺-channels. IBMX, a phosphodiesterase inhibitor, may be usedto raise c-AMP. Other agents, such as theophylline, may also be used toraise c-AMP. Agents, such as dibutyrl c-AMP, may be used to increasec-AMP directly. These agents maximally increase potassium conductanceand secretion, permitting the identification of reduced potassiumsecretion and conductance associated with at-risk or pre-canceroustissue.

[0082] Another such method employs measurements with a series of varyingKCl concentrations in contact with the colonic mucosa, such as 10, 20,40, and 80 mM KCl. Electrodes containing 10 μM-1 mM amiloride in the ECMare used to measure TEP and impedance, both in the presence and absenceof K⁺-channel blockers, such as 20 mM TEA (tetraethyl ammonium) and 5 mMbarium. The TEP is lower than normal in the at-risk and pre-canceroustissue. The impedance is lower than normal in the cancerous tissue. Intransitional tissue or tissue adjacent to developing cancer, impedancemay be higher than normal.

[0083] Chloride: Similar to the methods for sodium and potassiumdescribed above, chloride conductance can be used to determine abnormalpre-cancerous and cancerous tissue. Chloride conductance occurs mainlyat the base of the crypt (or deep) in normal epithelium. In canceroustissue, the epithelial cells closer to the surface of the crypt becomemore conductive to chloride, albeit at a lower level of conductance thanobserved in the base. The ratio of chloride conductance between thesurface and the base, as estimated from impedance measurements, can beused to characterize colonic tissue as either normal, at-risk,pre-cancerous, or cancerous. Specifically, at-risk and pre-cancerousepithelium exhibits an overall decrease in chloride conductance, with anincrease in the surface/base ratio. As the tissue progresses tocancerous, the overall chloride conductance increases and is accompaniedby increased Cl⁻ secretion. The surface/base ratio may become lessdiscriminatory, however, because normal epithelial morphology is lost ina malignant tumor.

[0084] As with potassium, chloride-dependent TEP is measured usingincreasing concentrations of Cl⁻. Measurements are made in the presenceof an ECM containing a sodium pump blocker agent, such as amiloride, inorder to negate the contribution of electrogenic Na⁺ transport, andagents, such as bethanacol, forskolin, and IBMX to maximally open Cl⁻conduction channels in the surface cells of the colon. The wells haveCl— concentrations varying between 15 and 120 mM, while maintaining theconcentrations of Na and K ions and keeping osmolality constant. Inat-risk and pre-cancerous tissue, the Cl⁻ is reduced. Additionally, theTEP is lower than normal. In cancerous tissue, the basal Cl⁻ secretionand Cl⁻ conductance is increased.

[0085] Drug Provocation: In addition to the ionic manipulationsdescribed above, the colon responds to a number of different hormones,growth factors, and diets by changing the ion transport characteristicsof the epithelium. For example, aldosterone (a mineralocorticoid) anddexamethasone (a glucocorticoid) both increase electrogenic sodiumabsorption and potassium secretion in the colon. In normal colon, sodiumconductance is increased in surface cells and the epitheliumhyperpolarizes, or becomes more negative in the lumen. Potassiumconductance increases in the deeper cells. In at-risk, pre-cancerous, orcancerous tissue, however, this response is significantly different. Thehyperpolarization and increase in sodium conductance is markedlydiminished. The increase in the potassium conductance in the basal cellsof the crypt is much less than occurs in normal colon. Thus, agents andtreatments that affect the ion transport characteristics of theepithelium may be used to enhance differences between normal andabnormal colon tissue in impedance measurements and/or othermeasurements of the electrical characteristics. A high-potassium,low-sodium diet will produce similar effects in a normal bowel. Otheragents may be administered directly to the surface of the bowel andproduce similar effects in normal epithelium. Carbenoxolone, forexample, when administered rectally, increases TEP in normal bowel, buthas a lesser effect on pre-cancerous or cancerous tissue. It causes anincrease in TEP because it inactivates 11β-HSD (11-beta hydroxysteroiddehydrogenase). Cortisol has mineralocorticoid effects on the bowel andincreases electrogenic sodium absorption and therefore increases TEP innormal but not in abnormal or cancerous bowel epithelium.

[0086]FIG. 5A demonstrates the short circuit current of human colonicepithelium ex-vivo. The figure demonstrates the time course along thex-axis while varying the potassium gradient across the tissue. Thepotassium permeability of the apical membrane of human colonic mucosa(P^(K) _(a)) was determined in surgical specimens of controls andgrossly normal-appearing mucosa obtained 10-30 cm proximal to colorectaladenocarcinomas. The mucosa was mounted in Ussing chambers and thebasolateral membrane resistance and voltage were nullified by elevatingthe K⁺ in the serosal bathing solution. The apical sodium (Na⁺)conductance was blocked with 0.1 mM amiloride. This protocol reduces theequivalent circuit model of the epithelium to an apical membraneconductance and electromotive force in parallel with the paracellularpathway as has been verified by microelectrode studies. Increasingserosal K⁺ caused the I_(sc) to become negative (−140 μA/cm²) in normalcolon after which 30 mM mucosal TEA caused an abrupt increase in Isccorresponding to block of apical K⁺ channels. In cancer-bearing colonthe reduction in Isc is to −65 μA/cm². The serosal bath was remainedconstant at 125 mM [K].

[0087]FIG. 5B demonstrates that ΔI_(sc), determined with respect to theI_(sc) at 125 mM mucosal K, is a linear function of the concentrationgradient, Δ[K]. Because the voltage across the apical membrane is zerounder these conditions and the paracellular pathway is nonselective, theP^(K) _(a) (apical potassium permeability) can be calculated using theFick equation—i.e., I_(sc)=F×P^(K) _(a)Δ[K] where F is the Faradayconstant and Δ[K] is the concentration difference for K⁺ across theepithelium. FIG. 5b demonstrates mean ± sem values for I_(sc) in bothnormal and premalignant human distal colon. The apical K⁺ permeabilityof controls was 9.34×10⁻⁶ cm/sec and this was significantly reduced by50% in premalignant human mucosa to 4.45×10⁻⁶ cm/sec. P^(K) _(a) couldalso be calculated for the change in I_(sc) when the K⁺ channels wereblocked with TEA, assuming complete block. This resulted somewhat lowervalues of 6.4×10⁻⁶ cm/sec and 3.8×10⁻⁶ cm/sec corresponding to a 40%reduction in P^(K) _(a).

[0088] These observations show that there is a field change in the K⁺permeability and conductance of human colon, during the development ofcancer. Impedance measurements, DC measurement using electrodes withdifferent potassium gradients together with specific drugs, such asamiloride to block the contributions of electrogenic Na⁺ transport tothe electrical properties of the bowel are useful to diagnose coloncancer.

[0089]FIG. 6 is a photomicrograph which illustrates some of thecomplexities associated with electrophysiological and histologicalalterations that occur in the development of colonic cancer. The canceris a 10 mm in diameter, invasive and an ulcerated lesion that couldeasily be missed at colonoscopy (because it is a depressed lesion). Thecancer is depolarized to 0 mV with a much higher conductance than thesurrounding epithelium. The surrounding or adjacent epithelium is alsodepolarized at about −20 mV but has a higher impedance than the canceror normal epithelium. Note that the darker layer, the epithelium (e), ison the top surface. This is one cell layer thick, but form crypts, likeinverted test tubes with proliferation and secretory function at thebase and differentiated cells and absorptive function at the mouth. Theinferior layer (m) is the muscle layer of the bowel. This small tumorhas already invaded the muscle layer. More distant epithelium is alsodepolarized but to a lesser degree at −40 mV. Potassium conductance isdecreased in this morphologically normal-appearing epithelium. Chloridesecretion is also decreased compared to the tumor, which may activelysecrete chloride. The sodium conductance, G_(Na), is decreased and theNa/H exchanger is upregulated. The colonic mucosa tends to be thickenedwith elongated crypts in the region of the developing cancer (adjacentzone). Most of the impedance resides in the epithelial layer, andtherefore a higher impedance below 750 μm indicates an epithelialthickening associated with cancer. Recognizing the electrophysiologicalpattern enables a diagnosis of cancer to be made, i.e. anelectrophysiological virtual biopsy.

[0090]FIG. 7 demonstrates measurements of surface mucosal (epithelial)electropotential referenced to the serosal surface on a freshly excisedspecimen of pelvic colon and rectum from a 45-year-old male with anulcerated rectal carcinoma. Following resection the specimen wasimmediately opened in a longitudinal direction and surfaceelectropotential measurements were made using different ECMs. Followingexcision there is usually a decrease in the electropotential(“run-down”) of 5-10 mV in the first 5-10 minutes, although the relativeelectropotential differences from region to region remain similar.

[0091] The “starburst” at the lower end of the figure, 2-3 cms from theanal canal and 5 cms from the anal verge has an electropotential of +10mV measured over the surface of the tumor (left hand column “NormosolRingers's”). Normosol Ringer's is a physiological saline solutioncontaining approximately 5 mM K⁺. The normal mucosal surfaceelectropotential is −50 to −70 mV in the rectum. As measurement aretaken some distance from the tumor the bowel remains depolarized even upto 20 cm from the edge of the tumor where readings of −40 to −45 mV areobserved. This region is depolarized relative to normal colon wherelevels of less than −50 mV are observed.

[0092] When electropotential measurements are made in normal colon usingan ECM with a higher K⁺ concentration an increase in electropotential(increased positivity) of 20 mV or greater is frequently observed. Thisis because the normal colon is selectively permeable to K⁺ and theincreased ECM K⁺ concentration sets up a diffusion potential for K⁺across the ion-selective conductance pathways. In the cancer bearingcolon K+ conductance decreases in the region of the developing tumor aswell as some distance from it (“field-cancerization”). Up to 5 cm fromthe developing cancer there is essentially no change in the measuredelectropotential when the ECM is changed from 5 to 30 mM K⁺ (change fromleft column (“Normosol Ringer's”) to middle column “30 mM KCl” infigure). Up to 20 cm from the tumor the change in electropotential doesnot exceed 15 mV (−45 to −30 mv) 20 cms from the edge of the tumor. Afurther increase in the K⁺ concentration of the ECM results in smallincreases in positivity away from the tumor or an anomalous decrease inpositivity near or at the tumor, suggesting that a diffusion gradientfor a different ion (other than K⁺) is set-up in the vicinity of thetumor.

[0093] Depolarization in combination with altered K⁺ conductance andpermeability may be used to diagnose the presence of cancer or increasedrisk of cancer. Altered K⁺ conductance is observed before tumors developin the bowel. Combination with simultaneous impedance measurementsincreases diagnostic accuracy.

EXAMPLE 2

[0094] Breast Cancer

[0095] As mentioned above, impedance and DC electrical potential havebeen used separately at the skin's surface to diagnose breast cancer. Inthe current invention, the impedance characteristics of the overlyingskin or epithelium are measured and factored in to the diagnosticinterpretation of the data. For example the surface potential may bemore positive (or less negative) than the reference site because ofincreased conductance of the overlying skin, rather than because of anunderlying tumor.

[0096] The electrodes are placed over the suspicious region and thepassive DC potential is measured. Then AC impedance measurements aremade as discussed below. The variable impedance properties of theoverlying skin may attenuate or increase the measured DC surfaceelectropotentials. Alternatively, impedance measurements at differentfrequencies may initially include a superimposed continuous sine wave ontop of an applied DC voltage. Phase, DC voltage and AC voltage will bemeasured. The resistance of the skin or other epithelium at AC and adifferent resistance at DC are measured. Under DC conditions since thereis no phase shift we are able to measure the transepithelial potentialat the surface. The capacitive properties of the skin allow theunderlying breast epithelial and tumor potential to be measured at theskin surface.

[0097] Once the ECM results in “wetting” of the skin surface there ispseudo-exponential decay in the skin surface potential using the abovereferenced approach. Ions in the ECM diffuse through the skin and makeit more conductive, particularly because of changes in the skin parallelresistance. The time constant for this decay is inversely proportionalto the concentration and ionic strength of the gel. Once the skin isrendered more conductive by the ECM the capacitive coupling of thesurface to the underlying potential of the tumor or the surroundingepithelium is lost so that the measured potential now reflects an offsetand diffusion potential at the electrode-ECM-skin interfaces.

[0098] The use of pharmacological and/or hormonal agents, however, incombination with both impedance and DC electrical potential, provides aneven more effective method for detecting abnormal pre-cancerous orcancerous breast tissue. Breast cancer develops within a background ofdisordered proliferation, which primarily affects the terminal ductallobular units (TDLUs). The TDLUs are lined by epithelial cells, whichmaintain a TEP. In regions of up-regulated proliferation, the ducts aredepolarized. The depolarization of ducts under the skin surface iscapacitively coupled with the overlying skin, which results in skindepolarization. When a tumor develops in a region of up-regulatedproliferation the overlying breast skin becomes further depolarizedcompared with other regions of the breast and the impedance of thecancerous breast tissue decreases. Electrophysiological responses in TEPand impedance change under the influence of hormones and menstrualcycle.

[0099] For example, the electrophysiological response of breast tissueto 17-β-estradiol has been observed to be different in pre-cancerous orcancerous tissue than in normal breast tissue. In one method of presentinvention, estradiol is introduced directly into the duct orsystematically following sublingual administration of 17-β-gestradiol (4mg). This agent produces a rapid response, which peaks at approximately20 minutes. The electrophysiological response depends, in part, on thestage of the patient's menstrual cycle, as well as the condition of thebreast tissue. Specifically, in normal breast tissue, a rise in TEP willoccur during the follicular (or early) phase. In pre-cancerous orcancerous tissue, this response is abrogated. Post-menopausal women atrisk for breast cancer may have an exaggerated TEP response to estradiolbecause of up-regulated estrogen receptors on epithelial cell surfaces.

[0100]FIG. 8 demonstrates the effect of varying the ionic content of thebathing Ringers solution on transepithelial conductance. The humanbreast epithelial cells were grown as monolayers on Millipore filtersand grew to confluence in 7 to 10 days. The epithelia were then mountedin modified Ussing chambers and the DC conductances were measured usinga voltage clamp. The conductance was measured by passing a 2 μA currentpulse for 200 milliseconds and measuring the DC voltage response andcalculating the transepithelial conductance (y-axis), and plotting itagainst time (x-axis). The conductance was measured first in standardRinger solution, then in a sodium-free Ringer, then returned to standardRinger, then in a potassium-free Ringer and finally returning tostandard Ringer solution while maintaining normal osmolality during thestudies.

[0101] The upper plot (filled squares and solid line) demonstrates theconductance of benign human breast epithelia grown as a monolayer. Theconductance is higher in the benign epithelial cells. The Na⁺ and K⁺components of conductance are approximately, 10 and 5 mS.cm⁻²respectively.

[0102] The lower plot (filled circles and dotted line) demonstrates theconductance of malignant human breast epithelia grown as a monolayer.The conductance is significantly lower in the malignant epithelialcells. The Na⁺ and K⁺ components of conductance are approximately, 4 and1 mS.cm⁻² respectively.

[0103] In malignant tumors as opposed to monolayers of malignantepithelial cells the tight junction between cells break down and thetumor becomes more conductive than either benign or malignant epithelialmonolayers. This observation may be exploited in the diagnosis of breastcancer. The lower conductance of the epithelium around a developingtumor, together with a region of high conductance at the site of themalignancy, may be used to more accurately diagnose breast cancer. Usingelectrodes with ECMs with different ionic composition will permit thespecific ionic conductances to be used in cancer diagnosis. For examplea high conductance region with a surrounding area of low K-conductanceis indicative of breast cancer, A high conductance area with asurrounding region of normal conductance may be more indicative offibrocystic disease (a benign process).

[0104]FIG. 9 demonstrates measurements of cell membrane potential (Ψ) inhuman breast epithelial cells. Measurements were made using apotentiometric fluorescent probe, and ratiometric measurements, whichare calibrated using valinomycin and K³⁰-gradients. Ψs were measured inthe presence (closed circles) and absence (open circles) of estradiol(the active metabolite of estrogen). Each symbol is the meanmeasurement. The upper error bar is the standard error of the mean, andthe lower error bar is the 95% confidence level for the observations.The addition of estrogen to cultured breast epithelial cells results inan instantaneous increase in Ψ (data not shown) as well astransepithelial potential see FIG. 10. Transepithelial potential (V_(T))of an epithelium is the sum of the apical (luminal) cell membranepotential (V_(A)) and the basolateral (abluminal) cell membranepotential (V_(BL)). Therefore V_(T)=V_(A)+V_(BL)(changes in V_(A) andV_(BL) will therefore alter V_(T) or transepithelial potential).

[0105]FIG. 9 demonstrates that benign breast epithelial cells have a Ψof approximately −50 mV in the absence of estradiol and −70 mV whenestradiol is added to the culture media. Malignant and transformed cellshave a Ψ of between −31 and −35 mV in the absence of estradiol andapproximately 50 mV when estradiol is present in the culture medium.

[0106] The difference in the electrical properties may be exploited todiagnose breast cancer in vivo. Surface electropotential measurementsare a combination of the transepithelial potential, tumor potential andoverlying skin potential. Physiological doses of estradiol may beadministered to the patient to increase Ψ and the sustained effect ofestradiol results in an increase in transepithelial potential and tumorpotential measured as an increase in surface electropotential. Theincrease following sustained exposure (as opposed to the instantaneousresponse) is less in malignant than benign breast tissue.

[0107] It should be noted that the instantaneous response, illustratedin FIG. 10, is greater in malignant epithelia, whereas the chronic orsustained exposure to estradiol results in a lower increase in TEP(transepithelial electropotential) in malignant cells. Concurrentmeasurement of surface electropotential and impedance allow the moreaccurate diagnosis of cancer. FIG. 10 demonstrates the instantaneouseffect of increasing doses of estradiol on the transepithelial potential(TEP) of benign and malignant human breast epithelial cells. The cellswere grown as monolayers on Millipore filters and grew to confluence in7 to 10 days. The epithelia were then mounted in modified Ussingchambers and the TEP was measured using a voltage clamp. Increasingdoses of estradiol between 0 and 0.8 μM were added (x-axis). Thetransepithelial potential was measured after each addition and the TEPwas measured (y-axis).

[0108] The different dose response is apparent for benign and malignantepithelia. Malignant epithelia have a lower TEP but undergo aninstantaneous increase in TEP of approximately 9 mV (becomes moreelectronegative and reaches a level of <6 mV) after exposure to only 0.1μM estradiol and then depolarize to approximately −2 mV with increasingdoses of estradiol up to about 0.5 μM. Benign epithelia have a lesserresponse to increasing doses of estradiol and do not peak until almost0.3 μM and then remain persistently elevated (higher electronegativity), unlike the malignant epithelia, with increasing doses ofestradiol.

[0109] This difference in dose response may be exploited to diagnosebreast cancer. Estradiol, or other estrogens, at a low dose will beadministered systemically, transcutaneously, or by other route. Theinstantaneous response of the surface electropotential and impedance maythen be used to diagnose breast cancer with improved accuracy overexisting diagnostic modalities using impedance or DC measurement alone.

[0110]FIG. 11 shows conductance measurements made at 2000 Hz at thesurface of the breast. At this frequency the influence of the overlyingskin impedance is less. There is still however some variable componentof skin impedance, which results in significant variability of themeasurement as evidenced by the overlapping error bars. Each symbolrepresents the median measurement with error bars the standard deviationof the mean.

[0111] Open symbols represent measurements made in patients with abiopsy proven malignancy, while closed symbols represent measurementsmade in patients whose subsequent biopsy proved to be a benign processsuch as fibrocystic disease. Malignant lesions are often associated withsurrounding breast epithelium that demonstrates up-regulatedproliferation. These regions (“adjacent region”) are depolarized and mayhave a lower conductance than either over the region of malignancy. Thisdecreased conductance may be because of decreased K⁺-conductance of theadjacent and pre-malignant epithelium as I have observed in human colon.

[0112] Each of the three groups of symbols represents measurements fromover a suspicious lesion or region, then the adjacent region, and thenover normal breast in an uninvolved quadrant of the breast. The firsttwo symbols (circles) in each of the three groups are impedancemeasurements where the median value is plotted against the left y-axisas conductance in mS.cm⁻². The second two symbols (squares) is thesurface electrical potential measured in mV and plotted against theright y-axis; each division equals 5 mV. The third two symbols(triangles) is the electrical index for benign and malignant lesions andis in arbitrary units and is derived from the conductance and surfacepotential measurement. It is immediately apparent that there is lessoverlap in the error bars (standard deviation of the mean). Thereforebreast cancer can be more accurately diagnosed using a combination ofsurface potential measurement and AC-impedance measurements. Furtherenhancements of this technique will involve the use of spaced electrodesto probe different depths of the breast, and the use of the hormones,drugs and other agents to differentially alter the impedance andtransepithelial potential from benign and malignant breast tissue, andmeasured at the skin surface. This will enable further improvements indiagnostic accuracy.

[0113] It should be understood that the surface potential measurement ofbreast tissue varies based on the position of the woman in her menstrualcycle. FIG. 12 illustrates this variance. This figure demonstrateselectropotential measurements taken over the surface of each breast at 8different locations with an array of 8 electrodes on each breastreferenced to an electrode on the skin of the upper abdomen.Measurements are taken with error bars equal to the standard error ofthe mean. Filled circles and filled squares represent the median valuefrom the left and right breast respectively. The vertical dotted line isthe first day of each menstrual cycle.

[0114] It can be seen that the median values for each breast tend totrack one another with lower values in the first half of menstrual cycle(follicular phase) and higher values in the latter part of cycle (lutealphase). Although the measured electrical values are not completelysuperimposed, because of other factors affecting the electropotential ofthe breast, it can be seen that the lowest levels of electropotentialare observed 8-10 days before menstruation and the rise to the highestlevels around the time of menstruation. This may be because estradiollevels are higher in the second part of menstrual cycle and directlyaffect breast surface electropotential.

[0115] The cyclical pattern of electropotential activity when a breastcancer or proliferative lesion is present is quite different. Similarlyhigher levels of surface electropotential are observed when measurementswere made in the afternoon compared with the morning. This informationcan be exploited in a number of different ways. Measurement of thesurface potential and impedance at different times during cycle enablesa more accurate diagnosis because of a different cyclical change insurface electropotential (i.e., the peak to peak change in potential isless over a malignant region, relative to normal areas of the breast)Secondly, estradiol or another agent that changes the electropotentialof the breast may be administered systemically, topically (transdermal),or by other means, and the drug or hormone-induced change in surfacepotential may be used as a provocative test to diagnose breast cancer.

[0116] In these ways breast cancer can be more accurately diagnosedusing a combination of surface potential measurement and AC-impedancemeasurements.

EXAMPLE 3

[0117] Nasopharyngeal Cancer

[0118] Using methods similar to those described with respect to coloncancer, it is possible to use pharmacological and hormonal agents toenhance electrophysiological alterations caused by nasopharyngealcancer. One exemplary method would be a nasopharyngeal probe that wouldinclude wells providing for varying concentrations of K⁺ and wouldperform simple DC measurements.

EXAMPLE 4

[0119] Prostate

[0120]FIG. 13 represent measurements of cell membrane potential (ψ) inhuman prostatic epithelial cells under different growth conditions. AVoltage-sensitive FRET (fluorescent energy transfer) probe was used forpotentiometric ratio measurements. It has two fluorescent components:CC₂-DMPE (Coumarin) and DISBAC₂(3) (Oxonol). The oxonol distributesitself on opposite sides of the cell membrane in a Nernstian manneraccording to the ψ. The voltage sensitive distribution of oxonol istransduced through a ratiometric fluorescence signal via the coumarinwhich is bound to the outside surface of the cell membrane therebyamplifying the fluorescence. Measurements were made using a fluorescencemicroscope and a digital imaging system. The ratio measurements arecalibrated using Gramicidin D to depolarize the cell membrane and thenvarying the external K⁺-concentration. The calibrated cell membranepotential in mV is depicted on the y-axis.

[0121] The filled bars indicates the ψ of exponentially growingprostatic epithelial cells before they reach confluence, whereas theopen bars depict the ψ of the cells once they reach confluence and cellgrowth slows. The first two bars demonstrate that prostatic epithelialcells are depolarized when rapidly growing and hyperpolarize by about 20mV when they reach confluence. The second pair of bars demonstrate thatexponentially growing cells are depolarized even in growth factordeprived culture conditions (stripped serum) and hyperpolarize less inthe absence of growth factors on reaching confluence. The final pair ofbars demonstrate that cells grown in the presence of the activemetabolite of testosterone, DHT (dihydroxytestosterone), are slightlyhyperpolarized during exponential growth, but depolarize on reachingconfluence.

[0122] These differences in cell membrane potential support the notionthat growth conditions of prostatic epithelia in vivo will likelyinfluence the cell membrane potential of prostatic epithelial cells.Cell membrane potential will influence the transepithelial potentialmeasured at the prostate surface. Alteration in the DC potentialmeasured trans-rectally in combination with impedance will be used todiagnose prostate cancer.

[0123]FIG. 14 demonstrates electropotential measurement made over theprostate of a patient with a normal prostate. The patient was undergoinga colonoscopy for screening, which was negative and had a normal PSA.The ECM (electroconductive medium) contained 5 mM K⁺ and physiologicalconcentrations of other electrolytes. The filled circles and solid linerepresent the measurement of surface electropotential (y-axis) startingat 1 cm from the anal verge to 8 cm along the anterior aspect of therectum (x-axis). The values increase from approximately −28 mV to −70 mVover the prostate and drop (depolarize) to approximately −52 mV over thetop of the prostate, and referenced to the bloodstream. When the ECM ischanged to a solution with the same osmolality, but with a K⁺concentration of 30 mM. The electropotential of the surface of therectal mucosa depolarizes to 30 mV (open circles joined by a dottedline). This indicates significant K⁺ permeability of the overlyingrectal mucosa. The higher region of electro-negativity over the prostateis consistently seen when the prostate is healthy.

[0124]FIG. 15 demonstrates measurement made in a patient with apreviously biopsied prostate cancer. The symbols and axes are the sameas in FIG. 14. The region of electro-negativity is lower over thecancerous prostate. In this case electropotential measurements ofbetween −26 and −27 mV were made over the cancerous lobe of the prostatei.e., 30 to 40 mV lower than observed over healthy prostate. When theECM was changed to a solution with a K⁺-concentration of 30 mM adepolarization of 8 −9 mV was observed, or about a third of thatobserved in healthy prostate. This indicates a decrease inK⁺-conductance and permeability of both the prostate and overlyingrectal mucosa.

[0125] These changes in the normal DC electrical profile of the prostatewill be used separately or in combination with AC impedance measurementsto diagnose prostate cancer. Identification of depolarization of theprostate relative to the higher polarity of the surrounding rectalmucosa together with decreased K⁺-conductance indicate the presence ofprostate cancer. Additional AC measurements with differently spacedelectrodes will permit probing of the underlying prostate to accuratelylocalize the site of the prostatic malignancy.

EXAMPLE 5

[0126] Chemopreventative and Therapeutic Use

[0127] In addition to the ionic, pharmacologic, and hormonal agentsdescribed above, the system and method of the present invention may beused with cancer preventative and therapeutic agents and treatments.Specifically, electrical measurement of altered structure and functionprovides a method for evaluating a patient's response to the drugswithout requiring a biopsy and without waiting for the cancer to furtherdevelop. Patients who respond to a given chemopreventative ortherapeutic agent would likely show restoration of epithelial functionto a more normal state. Patients who do not respond would show minimalchange or may even demonstrate progression to a more advanced stage ofthe disease. This system and method, thus, may be used by eitherclinicians or drug companies in assessing drug response or by cliniciansin monitoring the progress of a patient's disease and treatment, ormonitoring the process of carcinogenesis (cancer development), before anovert malignancy has fully developed.

[0128] Furthermore an understanding of the physiological basis of thealtered impedance permits more accurate diagnosis. For example impedancemay increase or decrease because of several factors. Increased stromaldensity of breast tissue may alter impedance. This is a non-specificchange, which may not have any bearing on the probability of malignancy.On the other hand a decrease in potassium permeability of the epitheliaaround a developing malignancy would increase impedance and would bemore likely associated with a developing cancer than a non-specificimpedance change. Additional information is obtained from my method byprobing the tissue to different depths using spaced voltage-sensingelectrodes. The use of electrophysiological, pharmacological andhormonal manipulations to alter impedance differentially in normalcompared to cancer-prone, pre-malignant or malignant tissue is anothersignificant difference, which enhances the diagnostic accuracy of myinvention over the above referenced one.

[0129] Other embodiments of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. It is intended that the specificationand examples be considered as exemplary only, with a true scope andspirit of the invention being indicated by the following claims.

1. A method for determining a condition of a region of epithelial tissuecomprising: placing at least two current-passing electrodes in contactwith or in proximity to a surface of the selected region of tissue;placing a plurality of measuring electrodes in contact with or inproximity to the surface of the selected region of tissue; establishinga signal between the current-passing electrodes; measuring, at one ormore of the measuring electrodes, an impedance associated with theestablished signal; introducing at least one agent into the selectedregion of tissue; measuring, at one or more of the measuring electrodes,an impedance associated with the established signal following theintroduction of the at least one agent; and determining the condition ofthe selected region of tissue based on the measured impedance, followingthe introduction of the at least one agent.
 2. The method of claim 1,wherein placing of the current-passing electrodes includes the step of:placing a probe in contact with or in proximity to the surface of theselected region of tissue, wherein the current-passing electrodes aresituated on the probe.
 3. The method of claim 1, wherein placing of themeasuring electrodes includes the step of: contacting a placing a probein contact with or in proximity to the surface of the selected region oftissue, wherein the measuring electrodes are situated on the probe. 4.The method of claim 1, wherein the step of determining the condition ofthe selected region of tissue includes: rating the tissue based on oneof the following ratings: normal, at-risk, pre-cancerous, or cancerous.5. A method for determining a condition of a region of epithelial tissuecomprising: placing at least one current-passing electrodes in contactwith or in proximity to a surface of the selected region of tissue;placing a plurality of measuring electrodes in contact with or inproximity to the surface of the selected region of tissue; establishinga signal between at least one current-passing electrode and at least oneof the measuring electrodes; measuring, at one or more of the measuringelectrodes, an impedance associated with the established signal;introducing at least one agent into the selected region of tissue;measuring, at one or more of the measuring electrodes, an impedanceassociated with the established signal following the introduction of theat least one agent; and determining the condition of the selected regionof tissue based on the measured impedance, following the introduction ofat least one agent.
 6. The method of claim 5, wherein placing of thecurrent-passing electrodes includes the step of: placing a probe incontact with or in proximity to the surface of the selected region oftissue, wherein the current-passing electrodes are situated on theprobe.
 7. The method of claim 5, wherein placing of the measuringelectrodes includes the step of: placing a probe in contact with or inproximity to the surface of the selected region of tissue, wherein themeasuring electrodes are situated on the probe.
 8. The method of claim5, wherein the step of determining the condition of the selected regionof tissue includes: rating the tissue based on one of the followingratings: normal, at-risk, pre-cancerous, or cancerous.
 9. A method formeasuring electrical properties of an epithelium, having a surface,using a combination of DC electrical measurements and impedancespectroscopy, comprising the steps of: measuring a DC potential at thesurface of the epithelium with a first and a second voltage-measuringelectrodes, wherein the voltage-measuring electrodes are associated witha reference point; placing a pair of current-passing electrodes incontact with or in proximity to the epithelium adjacent to thevoltage-measuring electrodes; detecting, via the voltage-sensingelectrodes, a resulting electrical signal at different points on theepithelial surface; applying an electrical signal to the pair ofcurrent-passing electrodes at a plurality of frequencies; monitoring theresulting electrical signal at the voltage-sensing electrodes associatedwith each of the plurality of frequencies; and determining the impedanceof the epithelium associated with the voltage-sensing electrodes basedon each of the plurality of frequencies and the resulting electricalsignal associated with each of the plurality of frequencies.
 10. Themethod of claim 9, wherein the reference point includes an intravenouselectrode or a skin electrode with low skin impedance.
 11. The method ofclaim 9, further including the steps of: positioning a depth electrode;detecting, via the depth electrode, a resulting electrical signal;monitoring the resulting electrical signal at the depth electrodeassociated with each of the plurality of frequencies; and determiningthe impedance of the epithelium associated with the depth electrode andone of the first or second voltage-sensing electrodes based on each ofthe plurality of frequencies and the resulting electrical signalassociated with each of the plurality of frequencies; and determining adifference in the impedance associated with the voltage-sensingelectrodes and the impedance associated with the depth electrode. 12.The method of claim 11, wherein the step of determining the differencein the impedance includes the step of: subtracting the impedanceassociated with the voltage-sensing electrodes from the impedanceassociated with the depth electrode and one of the first or the secondvoltage-sensing electrodes.
 13. The method of claim 11, wherein the stepof positioning the depth electrode includes the step of: placing thedepth electrode in contact with or in proximity to the surface of theepithelium at a different location than the voltage-sensing electrodes.14. The method of claim 9, wherein the first voltage-sensing electrodeincludes an electroconductive medium (ECM) having a first concentrationand the second voltage-sensing electrode includes the ECM having asecond concentration, further including the step of: estimating an ionicconductance of the epithelium based on the impedance associated with thevoltage-sensing electrodes and the ECM concentrations.
 15. The method ofclaim 9, wherein the first voltage-sensing electrode includes anelectroconductive medium (ECM) including a first agent and the secondvoltage-sensing electrode includes the ECM including a second agent,further including the step of: estimating an ionic conductance of theepithelium based on the impedance associated with the voltage-sensingelectrodes and the ECM agents.
 16. The method of claim 9, wherein theapplied electrical signal includes low and high frequency sinusoidalalternating currents.
 17. The method of claim 16, wherein the sinusoidalalternating currents are applied sequentially.
 18. The method of claim16, wherein the sinusoidal alternating currents are applied in acomposite form.
 19. The method of claim 9, wherein the resultingelectrical signal is a real part of a resulting potential differencemeasured over a current path across the current-passing electrodes. 20.The method of claim 9, wherein the plurality of frequencies falls withinthe range of 0.2-6000 Hz.
 21. The method of claim 9, wherein theplurality of frequencies falls within the range of 2 to 800 kHz.
 22. Themethod of claim 9, further including the step of: filtering theresulting electrical signal using a low-pass filter.
 23. The method ofclaim 9, further including the step of: reducing a DC component of theresulting signal using a band-pass filter.
 24. A method for measuringelectrical properties of an epithelium, having a surface, using acombination of DC electrical measurements and impedance spectroscopy,comprising the steps of: measuring a DC potential at the surface of theepithelium with a first and a second voltage-measuring electrodes,wherein the voltage-measuring electrodes are associated with a referencepoint; placing at least one current-passing electrodes in contact withor in proximity to the epithelium adjacent to the voltage-measuringelectrodes; detecting, via the voltage-measuring electrodes, a resultingelectrical signal at different points on the epithelial surface;applying an electrical signal to at least one of the current-passingelectrodes and at least one of the voltage measuring electrodes at aplurality of frequencies; monitoring the resulting electrical signal atthe voltage-measuring electrodes associated with each of the pluralityof frequencies; and determining the impedance of the epitheliumassociated with the voltage-measuring electrodes based on each of theplurality of frequencies and the resulting electrical signal associatedwith each of the plurality of frequencies.
 25. An apparatus fordetermining the condition of tissue of an epithelium using a combinationof surface DC electrical measurements and impedance spectroscopy, theapparatus comprising: a first pair of spaced electrodes for applying anelectrical signal to the epithelium; a second pair of spaced electrodesfor detecting a resulting electrical signal at different points on theepithelium; a means for applying the electrical signal to the first pairof electrodes at a plurality of frequencies; a means for measuring theresulting electrical signal at the second pair of electrodes at saidplurality of frequencies; a means for obtaining a measure of theimpedance of a part of the epithelium based on the resulting electricalsignal; and a means for obtaining a difference signal representing achange in impedance with frequency.
 26. The apparatus of claim 25,further comprising: one or more additional pairs of electrodes placed atdifferent locations than the first or second pairs of electrodes; and ameans for measuring a resulting electrical signal between the additionalpairs of electrodes; and a means for estimating an impedance fordifferent layers of the epithelium by subtracting the impedance valuesfrom two pairs of electrodes at each frequency.
 27. The apparatus ofclaim 25, wherein the second pair of electrodes includes different ECMconcentrations for estimating the specific ionic conductance of theepithelium.
 28. The apparatus of claim 25, wherein the second pair ofelectrodes includes different ECM agents for estimating the specificionic conductance of the epithelium.
 29. The apparatus of claim 25,wherein the means for applying the electric signal includes: a signalgenerator for producing simultaneously a plurality of low and highfrequency constant alternating current signals; and a connection meansfor supplying such signals to the first pair of electrodes.
 30. Theapparatus of claim 25, wherein the means for measuring the resultingelectrical signal includes: at least one adjustable gain voltageamplifier.
 31. The apparatus of claim 25, wherein the means forobtaining the difference signal includes a microprocessor computingdevice.
 32. The apparatus of claim 31, wherein the microprocessorcomputing device includes a low-pass filter.
 33. The apparatus of claim31, wherein the microprocessor computing device includes at least one ofa band-pass filter and a high pass filter.
 34. An apparatus fordiagnosing abnormal tissue using a combination of DC electricalmeasurements and impedance spectroscopy and variably spaced electrodeswith electroconductive medium having ionic compositions to monitor theeffects of various pharmacological agents and hormones, the apparatuscomprising: a first pair of spaced electrodes for applying an electricalsignal to an epithelium; at least one pair of voltage-sensing electrodeshaving electroconductive medium of different ionic composition tomeasure DC potential at different ionic concentrations; a second pair ofspaced electrodes for detecting a resulting electrical signal atdifferent points on the epithelium; a means for applying an agent; ameans for examining temporal changes in electrical properties of theepithelium responsive to the agent; a signal generating device forapplying electrical signal to the first pair of electrodes at aplurality of frequencies; a monitoring device for monitoring theresulting electrical signal at the second pair of electrodes at theplurality of frequencies; a computing device configured to perform thefollowing steps: obtaining the specific ionic conductance andpermeability of the epithelium based on the resulting electrical signalsat different electroconductive medium compositions; comparing theresulting electrical signals, specific ionic conductances andpermeabilities over an area of suspected abnormality with adjacent andmore distant regions of the epithelium; obtaining from the resultingelectrical signal a measure of impedance of a part of the epithelium atthe plurality of frequencies; comparing the impedance over the area ofsuspected abnormality with the adjacent and more distant regions of theepithelium; obtaining a difference signal representing an impedancechange with frequency; comparing the impedance over the area ofsuspected abnormality with the adjacent and more distant regions of theepithelium; subtracting the impedance measurement made between a firstset of electrodes from the impedance measurement made between a secondset of electrodes to estimate the impedance at different layers of theepithelium, wherein the distance between the first set of electrodes isless than the distance between the second set of electrodes; comparingthe impedance at different layers of the epithelium over the areasuspected of abnormality with adjacent and more distant regions of theepithelium; comparing electrical properties of the epithelium influencedby agents at different layers of the epithelium over the area suspectedof abnormality with adjacent and more distant regions of the epithelium;and comparing electrical properties of the epithelium at different timesinfluenced by variations in temporal factors at different layers of theepithelium over the area of suspected abnormality with adjacent and moredistant regions of the epithelium.
 35. A method for determining thecondition of a tissue, comprising: measuring a first DC potential of anarea of tissue using a first electroconductive medium; measuring asecond DC potential of said area of tissue using a secondelectroconductive medium that differs in its ionic concentration fromsaid first electroconductive medium; and comparing said first and secondmeasurements to determine the condition of said tissue.
 36. A method fordetermining the condition of a tissue, comprising: measuring a first DCpotential of an area of tissue using a first electroconductive medium;administering at least one agent; and measuring a second DC potential ofsaid area of tissue after said step of administering at least one agent;comparing said first and second measurements to determine the conditionof said tissue.
 37. A method for determining the condition of a tissue,comprising: measuring a first DC potential of an area of tissue using afirst electroconductive medium; allowing a period of time to pass;measuring a second DC potential of said area of tissue after said periodof time has passed; and comparing said first and second measurements todetermine the condition of said tissue.
 38. A method for determining thecondition of a tissue, comprising: measuring a first impedance of anarea of tissue using a first electroconductive medium; measuring asecond impedance of said area of tissue using a second electroconductivemedium that differs in its ionic concentration from said firstelectroconductive medium; and comparing said first and secondmeasurements to determine the condition of said tissue.
 39. A method fordetermining the condition of a tissue, comprising: measuring a firstimpedance of an area of tissue using a first electroconductive medium;administering at least one agent; measuring a second impedance of saidarea of tissue after said step of administering at least one agent; andcomparing said first and second measurements to determine the conditionof said tissue.
 40. A method for determining the condition of a tissue,comprising: measuring a first impedance of an area of tissue using afirst electroconductive medium; allowing a period of time to pass;measuring a second impedance of said area of tissue after said period oftime has passed; and comparing said first and second measurements todetermine the condition of said tissue.
 41. A method for determining thecondition of a tissue, comprising: measuring a first DC potential andimpedance of an area of tissue using a first electroconductive medium;measuring a second DC potential and impedance of said area of tissueusing a second electroconductive medium that differs in its ionicconcentration from said first electroconductive medium; and comparingsaid first and second measurements to determine the condition of saidtissue.
 42. A method for determining the condition of a tissue,comprising: measuring a first DC potential and impedance of an area oftissue using a first electroconductive medium; administering at leastone agent; measuring a second DC potential and impedance of said area oftissue after said step of administering at least one agent; andcomparing said first and second measurements to determine the conditionof said tissue.
 43. A method for determining the condition of a tissue,comprising: measuring a first DC potential and impedance of an area oftissue using a first electroconductive medium; allowing a period of timeto pass; measuring a second DC potential and impedance of said area oftissue after said period of time has passed; comparing said first andsecond measurements to determine the condition of said tissue.
 44. Anapparatus for determining the condition of a tissue, comprising: meansfor measuring at least one electrical property of an area of tissueusing a first electroconductive medium; means for measuring said atleast one electrical property of said area of tissue using a secondelectroconductive medium that differs in its ionic concentration fromsaid first electroconductive medium; and means for comparing saidmeasurements to determine the condition of said tissue.
 45. Theapparatus of claim 44, wherein said at least one electrical property isDC potential.
 46. The apparatus of claim 44, wherein said at least oneelectrical property is impedance.
 47. The apparatus of claim 44, furthercomprising means for measuring a plurality of electrical properties ofan area of tissue, wherein at least one electrical property is DCpotential and at least one electrical property is impedance.
 48. Anapparatus for determining the condition of a tissue, comprising: meansfor measuring at least one electrical property of an area of tissueusing a first electroconductive medium; at least one agent; means formeasuring said at least one electrical property of said area of tissueafter said agent is administered; and means for comparing saidmeasurements to determine the condition of said tissue.
 49. Theapparatus of claim 48, wherein said at least one electrical property isDC potential.
 50. The apparatus of claim 48, wherein said at least oneelectrical property is impedance.
 51. The apparatus of claim 48, furthercomprising means for measuring a plurality of electrical properties ofan area of tissue, wherein at least one electrical property is DCpotential and at least one electrical property is impedance.
 52. Anapparatus for determining the condition of a tissue, comprising: meansfor making a first measurement of at least one electrical property of anarea of tissue using a first electroconductive medium; means for makinga second measurement of said at least one electrical property of saidarea at a subsequent time to said first measurement; and means forcomparing said first and second measurements to determine the conditionof said tissue.
 53. The apparatus of claim 52, wherein said at least oneelectrical property is DC potential.
 54. The apparatus of claim 52,wherein said at least one electrical property is impedance.
 55. Theapparatus of claim 52, further comprising means for measuring aplurality of electrical properties of an area of tissue, wherein atleast one electrical property is DC potential and at least oneelectrical property is impedance.